The importance of this project is underscored by the emergence of several pathogenic bacterial strains that are resistant to all currently available antibiotics. One way to combat antibiotic resistance is to establish new enzymatic targets within resistant bacterial strains and design and synthesize small molecule inhibitors to target these enzymes. Based on bacterial genetic information, the meso-diaminopimelate (mDAP)/lysine biosynthetic pathway offers several potential anti-bacterial targets that have yet to be explored. Since there are no similar pathways in mammals, inhibitors that target one or more of the enzymes in this biosynthetic pathway will likely exhibit selective toxicity against only bacteria. It has been shown that deletion of the gene encoding for one of these enzymes, the dapE-encoded N-succinyl-L,L-diaminopimelic acid desuccinylase (DapE;EC 3.5.1.18), is lethal to Helicobacter pylori and Mycobacterium smegmatis. Even in the presence of lysine supplemented media H. pylori was unable to grow. Therefore, DapE9s are essential for cell growth and proliferation. A major limitation in developing a novel class of antibiotics that target DapE enzymes is the lack of X-ray crystallographic information. Recently, the PI in collaboration with Dr. Boguslaw Nocek at Argonne National Laboratory, solved the 2.0 and 2.3 E resolution structures of the mono and dinuclear Zn(II) DapE enzymes from the pathogenic bacterium Haemophilus influenzae. These structures provide the foundation needed to examine the binding of several new, potent inhibitors of DapE that were recently discovered by the PI, some of which have been shown to possess antimicrobial activity towards Escherichia coli. The specific aims of this proposal are: i) Discover novel DapE inhibitors by high throughput screening, ii) Analyze the determinants of substrate/inhibitor binding to DapE, and iii) Examine active site residues that are involved in substrate/inhibitor binding. Results obtained from these studies will identify new medicinal chemistry leads for optimization as well as mechanistic insight into the determinants of substrate recognition and binding. These data will provide insight into which functional groups are most important to increase inhibitor binding enthalpy to DapE. The most promising compounds discovered will be tested for antimicrobial properties in vivo against several pathogenic bacterial strains providing an avenue to translate newly discovered antimicrobial leads into the clinic. It is anticipated that the successful completion of the studies described in this proposal will provide benefits to healthcare and the general welfare of society given that multidrug-resistant organisms pose a serious and increasing treat to human health. PUBLIC HEALTH RELEVANCE: The importance of this project is underscored by the emergence of several pathogenic bacterial strains that are resistant to all currently available antibiotics. To address this problem, we propose to identify new medicinal chemistry leads for optimization and analyze the determinants of substrate/inhibitor binding to the dapE-encoded N- succinyl-L,L-diaminopimelic acid desuccinylase (DapE) from Haemophilus influenzae, a novel antimicrobial target that is apart of the lysine biosynthetic pathway in bacteria. The most promising compounds will be tested for antimicrobial properties in vivo against several pathogenic bacterial strains providing an avenue to translate newly discovered antimicrobial leads into the clinic.